Human tears are composed of water, electrolytes, small molecules such as carbohydrates and lipids, and a variety of proteins, several of which have an enzymic function. The principal proteins of tears include lysozyme (an enzyme which attacks bacterial cell walls), lactoferrin (an iron-sequestering and thus bacteriostatic protein with a free-radical scavenging function), secretory IgA antibodies, and lipid binding proteins of the lipocalin family (previously known as tear-specific prealbumin). A wide variety of other enzymes (e.g. glycosidases and lysosomal hydrolases) is also present but in much smaller amounts.
Present formulations of artificial tears act by replacing the volume of the tear film, but they can only do this while they remain in contact with the surface of the eye. A simple saline solution would remain in contact with the eye surface for only a few seconds and thus a viscosity improving component is required in the formulation. Such components presently used include hypromellose, hydroxyethylcellulose, carboxymethylcellulose, polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene glycol, dextran, hyaluronic acid, or carbomer 940 (polyacrylic acid). Such compounds act by mimicking the mucus present on the corneal surface and may interact with such mucus already present.
The presence of such mucus mimicking (mucomimetic) components can, in some instances, lead to symptoms of blurred vision due to slow mixing with tears, and irritation caused by the crystallisation of said components on lids and lashes.
Certain properties of natural human tears need to be understood for the better formulation of artificial tears to be used in treatment or alleviation of xe2x80x9cdry eyexe2x80x9d symptoms. Dry eye is a disorder of the tear film due to tear deficiency or excessive tear evaporation which causes damage to the exposed surface of the eye and is associated with symptoms of eye discomfort. Key physical properties involved in the function of fluid tears are surface tension and viscosity, both of which are thought to be important in spreading and maintenance of the pre-ocular tear film. Since dry eyes may be deficient in components which modulate these properties, any artificial tear formulation should contain these or suitable model materials having the same effect.
The viscosity of tears is shear-dependent and shows shear-thinningxe2x80x94i.e. the viscosity is high if measured at low speeds, but falls as the speed increases, and at high speeds (shear rates) approaches that of the solvent (Tiffany, 1991). This has advantages in the eye, in resisting gravitational drainage at low shears (eye open) but avoiding viscous dragging and epithelial damage at high shears (blinking).
It was thought at one time that mucus dissolved in the tears was, because of its known effect on viscosity and surface tension in mucus solutions, also responsible for these physical properties in tears. Having discovered that there is in fact little or no mucus in tears (Tiffany et al., 1996), or too little to have the observed effect, the inventors investigated other possible agents among the known constituents of human tears. It seemed possible that a small molecule such as a lipid might be responsible.
The vast majority of tear-associated lipid, such as that found bound to mucus collected from the eye (Moore and Tiffany, 1979) is from the Meibomian glands of the eyelid margin. Meibomian lipid is brought into contact with the aqueous fluid in formation of the pre-ocular film, but Stuchell et al. (1984) had reported a variety of polar lipids in tears, quite independent of this and of unknown tissue origin.
Of the known macromolecular components of tears, only a lipocalin protein, previously known as tear-specific prealbumin, is known to have any lipid-binding capacity, and has a broad lipid class specificity (Glasgow et al. 1995). Tear lipocalin is one member of a broad family of lipid-binding proteins called the lipocalins; many of these are small molecules (ca. 18-20 kDa) and include retinol-binding protein in serum and xcex2-lactoglobulin in bovine milk (Flower, 1996). The inventors surmised that extractable lipids might therefore be bound to tear lipocalin in such a way as to influence surface tension, but that other lipids might produce the same effect if all that is necessary is a protein-lipid interaction.
Thus the invention provides in one aspect, a formulation suitable for application to mammalian eyes, which formulation comprises a pharmaceutically acceptable, substantially isotonic aqueous electrolyte buffered at a pH of 5 to 8.5, containing a lipid binding protein such as tear lipocalin at a concentration of from 0.01 to 50 mg/ml, and a polar lipid selected from phospholipids and glycolipids, at a concentration of from 1 xcexcg/ml to 10 mg/ml.
In a preferred embodiment the lipid(s) and lipid binding protein are present as a soluble complex in the aqueous electrolyte. In a further embodiment, the formulation also contains one or more pharmaceutically acceptable preservative or bacteriostatic compounds, such as benzalkonium chloride, disodium EDTA or sodium perborate.
In a more preferred embodiment, the formulation, containing a lipid protein complex and suitable preservatives, shows shear-thinning and/or a surface tension of less than 47 mN/m.
A preferred range of cations for use in the aqueous electrolyte would be any of Na, K, Ca, or Mg. A preferred range of anions would be any of Cl or HCO3, bearing in mind the preference for full solubility of the salt used.
The formulation should preferably be isotonic, or slightly hypotonic in order to combat any hypertonicity of tears caused by evaporation and/or disease. A preferable osmotic pressure for the solution would be 200-500 mOsmol/kg
Preferred protein choice(s) for inclusion in the formulation would be from any of lysozyme, lactoferrin, IgA, xcex2-lactoglobulin or lipocalin, or any other protein, preferably lipid-binding, capable of reducing the surface tension and facilitating shear-thinning when present in the formulation, described above. Optionally recombinant human tear-specific prealbumin would be used. This could be produced by methods well known in the art using the cloned gene for human tear lipocalin (Lassagne and Gachon, 1993). A process for the production of human lysozyme from a synthetic gene is disclosed in EP0181634. A preferable range of protein concentration in the formulation would be 0.9 to 1.7 mg/ml.
The use of proteins from other species or manufactured using recombinant techniques in a formulation as above, raises the possibility of toxic, allergenic and/or immunogenic effects. Therefore, the formulation of the invention would preferably contain a non-toxic, non-immunogenic, hypoallergenic protein as the protein component.
Preferred choice(s) of lipid component(s) would be any polar member(s) of any of the phospholipid, glycolipid and sphingolipid classes capable of reducing the surface tension and facilitating shear-thinning when present in the formulation as previously described. Such classes of lipid include the sphingosides, ceramides and gangliosides, and others as described in Gunstone and Herslxc3x6f, (1992). The lipid may have limited solubility. A preferred concentration range for such a lipid component would be 50 to 200 xcexcg/ml.
The formulation of the invention may also contain N-acetylcysteine as a mucolytic agent.
In one aspect the formulation of the invention will be applied to the eye in aqueous solution in the form of drops. These drops may be delivered from a single dose ampoule which may preferably be sterile and thus rendering bacteriostatic components of the formulation unnecessary. Alternatively, the drops may be delivered from a multi-dose bottle which may preferably comprise a device which extracts preservative from the formulation as it is delivered, such devices being known in the art. In another aspect, components of the invention may be delivered to the eye as a concentrated gel or similar vehicle which form dissolvable inserts that are placed beneath the eyelids.
In a general aspect, the invention aims to provide a substitute for natural tears that does not have just a mucus binding and/or lubricating capability. In addition or alternatively, the invention would reproduce the viscosity and surface tension properties of natural tears by way of its possible formulations, as detailed above. The formulation of the invention is useful in the treatment of eye irritations for example, those caused by environmental conditions such as atmospheric pollution or use of visual display units. The formulation may also be used with contact lenses or other ophthalmic products.